This invention relates to stabilization of biological cells, such as blood particles, for use in reference reagents employed in blood particle counting and sizing instruments. More particularly, this invention relates to stabilization of biological cells which have been treated for fixation with a polyaldehyde.
In the development of improved electronic blood particle counting and sizing equipment, such as the Coulter.RTM. type instruments, increased descrimination between particle types as determined by size distributions has required continuing improvement in reference solutions of known or well-established particle concentration and size distributions for use as controls to insure that the instruments are performing properly. While fresh human blood can be modified to prepare whole blood hematology reference controls, such suspensions have limited shelf life and begin to lose stability after approximately thirty days, even under chilled storage; such instability results in alteration of the mean cell volume (MCV) with consequent decrease in reliable particle discrimination.
In an attempt to stabilize a liquid blood control standard by actual freezing with diol or triol additives, U.S. Pat. No. 4,199,471 teaches a treatment in which red cells are successively treated with aldehyde, followed by slow admixture with diol or triol, and retention in a buffered solution. The emphasis of this patent upon admixture of the diol or triol, in contrast to reaction, is illustrated by the use of formaldehyde as the preferred aldehyde in the examples of the disclosed treatment.
In another method disclosed in U.S. Pat. No. 4,160,644, a platelet reference control is stabilized by addition of a minor amount of solid polyethylene glycol either to the platelet suspension or to the diluent for the platelet suspension, whereby time and agitation stabilization are both achieved; liquid polyethylene glycol, having lower molecular weight, was found to be ineffective.
The use of separate reference control reagents for different particle types has been more difficult than use of whole blood controls. For example, any system for platelet counting that distinguishes human platelets from other cells in the blood sample based on the characteristic size range and volume distribution of platelets requires that the control material used must also closely simulate the same platelet size range and volume distribution. Any reference control containing platelets or simulated platelets that have a narrow size distribution range would not be useful to determine whether the size distribution limits were correctly set. To this end, the reference control material must have a mean size that lies between the upper and lower size limits and should have a volume distribution histogram that closely approximates the log-normal distribution of fresh human platelets.
Many types of technology have been applied to this problem to produce a product having suitable size, shape, and volume and electrical resistivity, as well as temperature and time stability, uniform particle dispersion and microbiologic inhibition. The principal types of particles investigated previously have been synthetic particles, such as polystyrene latex, non-animal yeast and pollen cells, chemically-fixed human or other mammalian platelets and chemically-fixed animal cells of other types. Synthetic particles may be manufactured to very close mean volume and size distribution specifications, but smooth, stable and uniform suspensions are very difficult to prepare. In addition, the spherical shape of these particles presents a different electronic "shape" to a Coulter Counter.RTM., and these volumes must be "corrected" for the difference in shape by an arbitrary shape factor. Introduction of an undefined mathematical factor into the control process is undesirable. Pollens and yeasts share these same disadvantages and, in addition, suffer from lack of uniformity from batch to batch and from lack of availability. Human and other animal platelets are rather difficult and expensive to collect and prepare. The preservation processes now in commercial use cause significant shrinkage of platelets which upsets the normal volume population distribution; and the platelets are adversely affected on aging from deterioration, thus limiting the usefulness of a given lot of platelet control.
In a method disclosed in U.S. Pat. No. 3,553,310, biological cells, such as goat erythrocyes and microbial cells, are stabilized by chemical fixation in reaction with aldehydes, particularly glutaraldehyde, to form particles which are preserved and stabilized against lysis. Although aldehyde fixation is desirable in terms of particle stability, such fixation also causes cross-linking that may occur between particles which leads to undesirable aggluttination.
Typically, only one of the aldehyde groups of glutaraldehye (1,5-pentandialdehyde, OHCCH.sub.2 CH.sub.2 CH.sub.2 CHO) reacts with a biological cell, for example an erythrocyte (RBC) membrane protein molecule, leaving the other aldehyde group free to cross-link with other adjacent functional groups on the same cell or on other cells. ##STR1##
Other proposed mechanisms for this reaction involve formation of a glutaraldehyde dimer, containing a C.dbd.C with an active beta hydrogen. Condensation at this position leaves both aldehyde groups free. ##STR2##
By either mechanism, pseudo-hemagglutination (i.e. cross-linking between adjacent cells) results, causing cell clumps that settle very rapidly and tend to stay matted on the container bottom. Although this process is a slow one, it seriously affects tha stability and usefulness of any analog control made in this manner.